Methods for producing biological substances in pigment-deficient mutants of bacillus cells

ABSTRACT

The present invention relates to methods of producing a heterologous biological substance, comprising: (a) cultivating a mutant of a parent  Bacillus  cell under conditions conducive for the production of the heterologous biological substance, wherein (i) the mutant cell comprises a first nucleic acid sequence directing synthesis of the heterologous biological substance and a second nucleic acid sequence comprising a modification of at least one of the genes cypX and yvmC, which are involved in the production of a red pigment, and (ii) the mutant cell is deficient in the production of the red pigment compared to the parent  Bacillus  cell when cultivated under the same conditions; and (b) recovering the heterologous biological substance from the cultivation medium. The present invention also relates to mutants of  Bacillus  cells and methods for producing the mutants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/331,323, filed Dec. 9, 2008, now U.S. Pat. No. 7,666,625, which is acontinuation of U.S. application Ser. No. 10/627,124, filed Jul. 25,2003, now U.S. Pat. No. 7,476,516, which claims the benefit of U.S.Provisional Application No. 60/398,853, filed Jul. 26, 2002, whichapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of producing heterologousbiological substances in pigment-deficient Bacillus mutant cells,methods of obtaining the pigment-deficient Bacillus mutant cells, andthe pigment-deficient Bacillus mutant cells.

2. Description of the Related Art

The pulcherrimins are reddish pigments resulting from chelation offerric ions by pulcherriminic acid. The pulcherrimins consist ofsubstituted pyrazine rings with isobutyl groups bound to positions 2 and5, but differ slightly in other structural details (Kuffer et al., 1967,Archiv für Mikrobiologic 56: 9-21).

MacDonald, 1967, Canadian Journal of Microbiology 13: 17-20, hasdescribed the isolation of pulcherrimin from Bacillus cereus andBacillus subtilis and its conversion to the free acid pulcherriminicacid. Uffen and Canale-Parola, 1972, Journal of Bacteriology 111: 86-93,describe the synthesis of pulcherriminic acid by Bacillus subtilis.

Bacilli are well established as host cell systems for the production ofnative and recombinant proteins or other biological substances. However,Bacillus hosts with the desirable traits of increased protein expressionand secretion may not necessarily have the most desirablecharacteristics for successful fermentation, recovery, and purificationof biological substances produced by the cells. These processes may notbe optimal because of pigment formation requiring removal during therecovery and purification of a biological substance of interest or thepigment may co-purify with the biological substance.

It is therefore an object of the present invention to provide improvedBacillus hosts which combine the capacity for expression of commercialquantities of a biological substance while being deficient in theproduction of red pigment.

SUMMARY OF THE INVENTION

The present invention relates to methods of producing a heterologousbiological substance, comprising:

(a) cultivating a mutant of a parent Bacillus cell under conditionssuitable for the production of the heterologous biological substance,wherein (i) the mutant cell comprises a first nucleic acid sequencedirecting synthesis of the heterologous biological substance and asecond nucleic acid sequence comprising a modification of at least oneof the genes cypX and yvmC, which are involved in the production of ared pigment, and (ii) the mutant cell is deficient in the production ofthe red pigment compared to the parent Bacillus cell when cultivatedunder the same conditions; and

(b) recovering the heterologous biological substance from thecultivation medium.

The present invention also relates to red pigment-deficient Bacillusmutant cells and methods for producing the red pigment-deficientBacillus mutant cells.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence of the cypX gene and itsdeduced amino acid sequence (SEQ ID NOS: 1 and 2, respectively).

FIGS. 2A and 2B show the genomic DNA sequence of the yvmA gene and itsdeduced amino acid sequence (SEQ ID NOS: 3 and 4, respectively).

FIG. 3 shows the genomic DNA sequence of the yvmB gene and its deducedamino acid sequence (SEQ ID NOS: 5 and 6, respectively).

FIGS. 4A and 4B show the genomic DNA sequence of the yvmC gene and itsdeduced amino acid sequence (SEQ ID NOS: 7 and 8, respectively).

FIG. 5 shows a restriction map of pMRT084.

FIG. 6 shows a restriction map of pMRT086.

FIG. 7 shows a restriction map of pMRT126.

FIG. 8 shows a restriction map of pMRT128.

FIG. 9 shows a restriction map of pMRT121.

FIG. 10 shows a restriction map of pMRT123.

FIG. 11 shows a restriction map of pMRT124.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of producing a heterologousbiological substance, comprising: (a) cultivating a mutant of a parentBacillus cell under conditions conducive for the production of theheterologous biological substance, wherein (i) the mutant cell comprisesa first nucleic acid sequence directing synthesis of the heterologousbiological substance and a second nucleic acid sequence comprising amodification of at least one of the genes cypX and yvmC, which areinvolved in the production of a red pigment, and (ii) the mutant cell isdeficient in the production of the red pigment compared to the parentBacillus cell when cultivated under the same conditions; and (b)recovering the heterologous biological substance from the cultivationmedium.

An advantage of the present invention is the elimination or reduction ofthe red pigment in a Bacillus fermentation broth. The elimination orreduction of the red pigment facilitates the recovery and purificationof a biological substance of interest.

In the methods of the present invention, the red pigment is believed tobe pulcherrimin because when a solid or liquid medium of a Bacillusculture is cultivated in the absence of ferric ions and then exposed toferric ions, the culture and/or cells becomes reddish in color.Moreover, the isolated pigment is soluble in alkali, insoluble in waterand organic solvents, and the UV-visible spectrum matches the previouslypublished spectrum for pulcherriminic acid (see, Canale-Parola, 1963,Archie für Mikrobiologie 46: 414-427). The term “pulcherrimin” isdefined herein as a ferric chelate or ferric salt of pulcherriminicacid. Pulcherriminic acid is the free acid of pulcherrimin, whichconsists of substituted pyrazine rings with isobutyl groups bound topositions 2 and 5, which may differ slightly in other structural details(Kuffer et al., 1967, supra).

The term “modification” is defined herein as an introduction,substitution, or removal of one or more nucleotides in the gene or aregulatory element required for the transcription or translationthereof; a gene disruption; gene conversion; a gene deletion; or randomor specific mutagenesis of at least one of the genes cypX and yvmC. Thedeletion of the cypX and/or yvmC gene(s) may be partial or complete.

The phrase “deficient in the production of the red pigment” is definedherein as a Bacillus mutant cell which produces no detectable redpigment, or, in the alternative, produces preferably at least about 25%less, more preferably at least about 50% less, even more preferably atleast about 75% less, and most preferably at least about 95% less redpigment compared to the parent Bacillus cell when cultivated under thesame conditions. The level of red pigment produced by a Bacillus mutantcell of the present invention may be determined using methods well knownin the art (see, for example, Kuffer et al., 1967, supra). However, thepresence or absence of the red pigment can be made visually bycentrifugation of the cell mass because the pigment adsorbs to thecells, whether the cultivation medium employed is a complex or minimalmedium. In a minimal medium, the red pigment can be observed in thesupernatant, but as the medium become more complex and colored from themedium components, the color of the components may mask the presence orabsence of the red pigment in a cell supernatant.

In the methods of the present invention, the parent Bacillus cell may bea wild-type Bacillus cell or a mutant thereof, which produces the redpigment. Bacillus cells useful in the practice of the present inventioninclude, but are not limited to, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis cells. In a preferred embodiment, the Bacillus cell is aBacillus amyloliquefaciens, Bacillus lentus, Bacillus licheniformis,Bacillus stearothermophilus or Bacillus subtilis cell. In a morepreferred embodiment, the parent Bacillus cell is a Bacillusamyloliquefaciens cell. In another more preferred embodiment, the parentBacillus cell is a Bacillus clausii cell. In another more preferredembodiment, the parent Bacillus cell is a Bacillus licheniformis cell.In another more preferred embodiment, the parent Bacillus cell is aBacillus subtilis cell.

The red pigment-deficient Bacillus mutant cell may be constructed byreducing or eliminating expression of at least one of the genes cypX andyvmC using methods well known in the art, for example, insertions,disruptions, replacements, or deletions. The portion of the gene to bemodified or inactivated may be, for example, the coding region or aregulatory element required for expression of the coding region. Anexample of such a regulatory or control sequence may be a promotersequence or a functional part thereof, i.e., a part which is sufficientfor affecting expression of the nucleic acid sequence. Other controlsequences for possible modification include, but are not limited to, aleader, propeptide sequence, signal sequence, transcription terminator,and transcriptional activator.

The Bacillus mutant cells may be constructed by gene deletion techniquesto eliminate or reduce the expression of at least one of the genes cypXand yvmC. Gene deletion techniques enable the partial or completeremoval of the gene(s) thereby eliminating their expression. In suchmethods, the deletion of the gene(s) may be accomplished by homologousrecombination using a plasmid that has been constructed to contiguouslycontain the 5′ and 3′ regions flanking the gene. The contiguous 5′ and3′ regions may be introduced into a Bacillus cell, for example, on atemperature-sensitive plasmid, such as pE194, in association with asecond selectable marker at a permissive temperature to allow theplasmid to become established in the cell. The cell is then shifted to anon-permissive temperature to select for cells that have the plasmidintegrated into the chromosome at one of the homologous flankingregions. Selection for integration of the plasmid is effected byselection for the second selectable marker. After integration, arecombination event at the second homologous flanking region isstimulated by shifting the cells to the permissive temperature forseveral generations without selection. The cells are plated to obtainsingle colonies and the colonies are examined for loss of bothselectable markers (see, for example, Perego, 1993, In A. L. Sonneshein,J. A. Hoch, and R. Losick, editors, Bacillus subtilis and OtherGram-Positive Bacteria, Chapter 42, American Society of Microbiology,Washington, D.C.).

The Bacillus mutant cells may also be constructed by introducing,substituting, or removing one or more nucleotides in the gene or aregulatory element required for the transcription or translationthereof. For example, nucleotides may be inserted or removed so as toresult in the introduction of a stop codon, the removal of the startcodon, or a frame-shift of the open reading frame. Such a modificationmay be accomplished by site-directed mutagenesis or PCR generatedmutagenesis in accordance with methods known in the art. See, forexample, Botstein and Shortle, 1985, Science 229: 4719; Lo et al., 1985,Proceedings of the National Academy of Sciences USA 81: 2285; Higuchi etal., 1988, Nucleic Acids Research 16: 7351; Shimada, 1996, Meth. Mol.Biol. 57: 157; Ho et al., 1989, Gene 77: 61; Horton et al., 1989, Gene77: 61; and Sarkar and Sommer, 1990, BioTechniques 8: 404.

The Bacillus mutant cells may also be constructed by gene disruptiontechniques by inserting into one or more of the genes responsible forthe production of the red pigment an integrative plasmid containing anucleic acid fragment homologous to the gene(s) which will create aduplication of the region of homology and incorporate vector DNA betweenthe duplicated regions. Such gene disruption can eliminate geneexpression if the inserted vector separates the promoter of the genefrom the coding region or interrupts the coding sequence such that anon-functional gene product results. A disrupting construct may besimply a selectable marker gene accompanied by 5′ and 3′ regionshomologous to the gene. The selectable marker enables identification oftransformants containing the disrupted gene.

The Bacillus mutant cells may also be constructed by the process of geneconversion (see, for example, Iglesias and Trautner, 1983, MolecularGeneral Genetics 189: 73-76). For example, in the gene conversionmethod, a nucleic acid sequence corresponding to the gene(s) ismutagenized in vitro to produce a defective nucleic acid sequence whichis then transformed into the parent Bacillus cell to produce a defectivegene. By homologous recombination, the defective nucleic acid sequencereplaces the endogenous gene. It may be desirable that the defectivegene or gene fragment also encodes a marker which may be used forselection of transformants containing the defective gene. For example,the defective gene may be introduced on a non-replicating ortemperature-sensitive plasmid in association with a selectable marker.Selection for integration of the plasmid is effected by selection forthe marker under conditions not permitting plasmid replication.Selection for a second recombination event leading to gene replacementis effected by examination of colonies for loss of the selectable markerand acquisition of the mutated gene (see, for example, Perego, 1993,supra). Alternatively, the defective nucleic acid sequence may containan insertion, substitution, or deletion of one or more nucleotides ofthe gene, as described below.

The Bacillus mutant cells may also be constructed by establishedanti-sense techniques using a nucleotide sequence complementary to thenucleic acid sequence of the gene (Parish and Stoker, 1997, FEMSMicrobiology Letters 154: 151-157). More specifically, expression of thegene by a Bacillus cell may be reduced or eliminated by introducing anucleotide sequence complementary to the nucleic acid sequence of thegene, which may be transcribed in the cell and is capable of hybridizingto the mRNA produced in the cell. Under conditions allowing thecomplementary anti-sense nucleotide sequence to hybridize to the mRNA,the amount of protein translated is thus reduced or eliminated.

The Bacillus mutant cells may be further constructed by random orspecific mutagenesis using methods well known in the art, including, butnot limited to, chemical mutagenesis (see, for example, Hopwood, TheIsolation of Mutants in Methods in Microbiology (J. R. Norris and D. W.Ribbons, eds.) pp 363-433, Academic Press, New York, 1970) andtransposition (see, for example, Youngman et al., 1983, Proc. Natl.Acad. Sci. USA 80: 2305-2309). Modification of the gene may be performedby subjecting the parent cell to mutagenesis and screening for mutantcells in which expression of the gene has been reduced or eliminated.The mutagenesis, which may be specific or random, may be performed, forexample, by use of a suitable physical or chemical mutagenizing agent,use of a suitable oligonucleotide, or subjecting the DNA sequence to PCRgenerated mutagenesis. Furthermore, the mutagenesis may be performed byuse of any combination of these mutagenizing methods.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),N-methyl-N′-nitrosoguanidine (NTG) O-methyl hydroxylamine, nitrous acid,ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, andnucleotide analogues. When such agents are used, the mutagenesis istypically performed by incubating the parent cell to be mutagenized inthe presence of the mutagenizing agent of choice under suitableconditions, and selecting for mutant cells exhibiting reduced or noexpression of the gene.

In the methods of the present invention, either the cypX or yvmC gene,or both, of a Bacillus cell involved in the production of the redpigment may be modified, as described herein. The cypX-yvmC operon wasidentified as a potential site involved in the formation of the redpigment by Bacillus ORFs microarray analysis according to the protocolof Berka et al., 2002, Molecular Microbiology 43: 1331-1345. It will beunderstood that the term “second nucleic acid sequence” may include oneor both of the genes cypX and yvmC.

In a preferred embodiment, cypX comprises a nucleic acid sequence havingat least 70%, preferably at least 75%, more preferably at least 80%,even more preferably at least 85%, most preferably at least 90%, andeven most preferably at least 95% homology to SEQ ID NO: 1. In a mostpreferred embodiment, cypX comprises the nucleic acid sequence of SEQ IDNO: 1. In another most preferred embodiment, cypX consists of thenucleic acid sequence of SEQ ID NO: 1.

In a preferred embodiment, yvmC comprises a nucleic acid sequence havingat least 70%, preferably at least 75%, more preferably at least 80%,even more preferably at least 85%, most preferably at least 90%, andeven most preferably at least 95% homology to SEQ ID NO: 7. In a mostpreferred embodiment, yvmC comprises the nucleic acid sequence of SEQ IDNO: 7. In another most preferred embodiment, yvmC consists of thenucleic acid sequence of SEQ ID NO: 7.

For purposes of the present invention, the degree of homology betweentwo nucleic acid sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

A nucleic acid sequence homologous or complementary to the nucleic acidsequences described herein, involved in the production of the redpigment, may be used from other microbial sources which produce the redpigment to modify the corresponding gene in the Bacillus strain ofchoice.

In a preferred embodiment, the modification of a gene involved in theproduction of a red pigment in the Bacillus mutant cell is unmarked witha selectable marker.

Removal of the selectable marker gene may be obtained by culturing themutants on a counter-selection medium. Where the selectable marker genecontains repeats flanking its 5′ and 3′ ends, the repeats willfacilitate the looping out of the selectable marker gene by homologousrecombination when the mutant cell is submitted to counter-selection.The selectable marker gene may also be removed by homologousrecombination by introducing into the mutant cell a nucleic acidfragment comprising 5′ and 3′ regions of the defective gene, but lackingthe selectable marker gene, followed by selecting on thecounter-selection medium. By homologous recombination, the defectivegene containing the selectable marker gene is replaced with the nucleicacid fragment lacking the selectable marker gene. Other methods known inthe art may also be used.

It will be understood that the methods of the present invention are notlimited to a particular order for obtaining the Bacillus mutant cell.The modification of the gene(s) involved in the production of the redpigment may be introduced into the parent cell at any step in theconstruction of the cell for the production of a biological substance.It is preferable that the Bacillus mutant cell has already been made redpigment-deficient prior to the introduction of a gene(s) directingsynthesis of a heterologous biological substance.

In a further aspect of the present invention, the mutants of Bacilluscells may additionally contain modifications, e.g., deletions ordisruptions, of other genes which may be detrimental to the production,recovery or application of a biological substance. In a preferredembodiment, the Bacillus cell is a protease-deficient cell. In a morepreferred embodiment, the Bacillus cell comprises a disruption ordeletion of aprE and nprE. In another preferred embodiment, the Bacilluscell does not produce spores. In another more preferred embodiment, theBacillus cell comprises a disruption or deletion of spoIIAC. In anotherpreferred embodiment, the Bacillus cell comprises a disruption ordeletion of one of the genes involved in the biosynthesis of surfactin,e.g., srfA, srfB, srfC, and srfD. See, for example, U.S. Pat. No.5,958,728. Other genes, e.g., the amyE gene, which are detrimental tothe production, recovery or application of a biological substance mayalso be disrupted or deleted.

In the methods of the present invention, the Bacillus mutant cellpreferably produces at least the same amount of the biological substanceas the corresponding parent Bacillus cell when cultured under identicalproduction conditions. In a more preferred embodiment, the mutant cellproduces at least about 25% more, preferably at least about 50% more,more preferably at least about 75% more, and most preferably at leastabout 100% more of the biological substance than the correspondingparent Bacillus cell when cultured under identical productionconditions.

The Bacillus mutant cells are cultivated in a nutrient medium suitablefor production of the heterologous biological substance using methodsknown in the art. For example, the cell may be cultivated by shake flaskcultivation, small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the biological substance to be expressedand/or isolated. The cultivation takes place in a suitable nutrientmedium comprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). The secreted biological substance can be recovered directlyfrom the medium.

The biological substances may be detected using methods known in the artthat are specific for the biological substances. These detection methodsmay include use of specific antibodies, high performance liquidchromatography, capillary chromatography, formation of an enzymeproduct, disappearance of an enzyme substrate, or SDS-PAGE. For example,an enzyme assay may be used to determine the activity of the enzyme.Procedures for determining enzyme activity are known in the art for manyenzymes (see, for example, D. Schomburg and M. Salzmann (eds.), EnzymeHandbook, Springer-Verlag, New York, 1990).

The resulting biological substance may be isolated by methods known inthe art. For example, a polypeptide of interest may be isolated from thecultivation medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray-drying, evaporation,or precipitation. The isolated polypeptide may then be further purifiedby a variety of procedures known in the art including, but not limitedto, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989). A metabolite of interest may be isolated from a cultivationmedium by, for example, extraction, precipitation, or differentialsolubility, or any method known in the art. The isolated metabolite maythen be further purified using methods suitable for metabolites.

The heterologous biological substance may be any biopolymer ormetabolite. The biological substance may be encoded by a single gene ora series of genes composing a biosynthetic or metabolic pathway. Thus,the term “first nucleic acid sequence directing synthesis of aheterologous biological substance” will be understood to encompass oneor more genes involved in the production of the biological substance.The term “heterologous biological substance” is defined herein as abiological substance which is not native to the host cell; a nativebiological substance in which structural modifications have been made toalter the native biological substance, e.g., the protein sequence of anative polypeptide; or a native biological substance whose expression isquantitatively altered as a result of a manipulation of the host cell byrecombinant DNA techniques, e.g., a stronger promoter.

In the methods of the present invention, the biopolymer may be anybiopolymer. The term “biopolymer” is defined herein as a chain (orpolymer) of identical, similar, or dissimilar subunits (monomers). Thebiopolymer may be, but is not limited to, a nucleic acid, polyamine,polyol, polypeptide (or polyamide), or polysaccharide.

In a preferred embodiment, the biopolymer is a polypeptide. Thepolypeptide may be any polypeptide having a biological activity ofinterest. The term “polypeptide” is not meant herein to refer to aspecific length of the encoded product and, therefore, encompassespeptides, oligopeptides, and proteins. The term “polypeptide” alsoencompasses two or more polypeptides combined to form the encodedproduct. Polypeptides also include hybrid polypeptides, which comprise acombination of partial or complete polypeptide sequences obtained fromat least two different polypeptides wherein one or more may beheterologous to the Bacillus cell. Polypeptides further includenaturally occurring allelic and engineered variations of theabove-mentioned polypeptides and hybrid polypeptides.

Preferably, the heterologous polypeptide is an antibody, antigen,antimicrobial peptide, enzyme, growth factor, hormone, immunodilator,neurotransmitter, receptor, reporter protein, structural protein, andtranscription factor.

In a preferred embodiment, the heterologous polypeptide is anoxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. Ina more preferred embodiment, the polypeptide is an alpha-glucosidase,aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, glucocerebrosidase, alpha-glucosidase, beta-glucosidase,invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolyticenzyme, peroxidase, phospholipase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, urokinase, orxylanase.

In a preferred embodiment, the biopolymer is a polysaccharide. Thepolysaccharide may be any polysaccharide, including, but not limited to,a mucopolysaccharide (e.g., heparin and hyaluronic acid) andnitrogen-containing polysaccharide (e.g., chitin). In a more preferredembodiment, the polysaccharide is hyaluronic acid.

In the methods of the present invention, the metabolite may be anymetabolite. The metabolite may be encoded by one or more genes. The term“metabolite” encompasses both primary and secondary metabolites. Primarymetabolites are products of primary or general metabolism of a cell,which are concerned with energy metabolism, growth, and structure.Secondary metabolites are products of secondary metabolism (see, forexample, R. B. Herbert, The Biosynthesis of Secondary Metabolites,Chapman and Hall, New York, 1981).

The primary metabolite may be, but is not limited to, an amino acid,fatty acid, nucleoside, nucleotide, sugar, triglyceride, or vitamin.

The secondary metabolite may be, but is not limited to, an alkaloid,coumarin, flavonoid, polyketide, quinine, steroid, or terpene. In apreferred embodiment, the secondary metabolite is an antibiotic,antifeedant, attractant, bacteriocide, fungicide, hormone, insecticide,or rodenticide.

In the methods of the present invention, the mutant of the Bacillus cellis a recombinant cell, comprising a nucleic acid sequence directingsynthesis of a heterologous biological substance, e.g., polypeptide,which is advantageously used in the recombinant production of thebiological substance. The cell is preferably transformed with a vectorcomprising the nucleic acid sequence directing synthesis of theheterologous biological substance followed by integration of the vectorinto the chromosome. “Transformation” means introducing a vectorcomprising the nucleic acid sequence into a host cell so that the vectoris maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector. Integration is generally considered to be anadvantage as the nucleic acid sequence is more likely to be stablymaintained in the cell. Integration of the vector into the chromosomeoccurs by homologous recombination, non-homologous recombination, ortransposition.

The nucleic acid sequence directing synthesis of a heterologousbiological substance may be obtained from any prokaryotic, eukaryotic,or other source, e.g., archaeabacteria. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the biological substance is produced by thesource or by a cell in which a gene from the source has been inserted.

In the methods of the present invention, the mutants of Bacillus cellsmay also be used for the recombinant production of biological substanceswhich are native to the Bacillus cell. The native biological substancemay be recombinantly produced by, for example, placing a gene(s)directing synthesis of the biological substance under the control of adifferent promoter to enhance expression of the substance, expeditingits export outside the cell by use of, for example, a signal sequence,or increasing the copy number of a gene directing synthesis of thebiological substance normally produced by the Bacillus cell. Thus, thepresent invention also encompasses, within the scope of the term“heterologous biological substances,” such recombinant production ofnative biological substances, to the extent that such expressioninvolves the use of genetic elements not native to the Bacillus cell, oruse of native elements which have been manipulated to function in amanner that do not normally occur in the host cell.

The techniques used to isolate or clone a nucleic acid sequencedirecting synthesis of a biological substance are known in the art andinclude isolation from genomic DNA, preparation from cDNA, or acombination thereof. The cloning of the nucleic acid sequences from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR). See, for example, Innis et al., 1990, PCRProtocols: A Guide to Methods and Application, Academic Press, New York.The cloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence directingsynthesis of the biological substance, insertion of the fragment into avector molecule, and incorporation of the recombinant vector into aBacillus cell where multiple copies or clones of the nucleic acidsequence will be replicated. The nucleic acid sequence may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

In the methods of the present invention, where the biological substanceis a heterologous polypeptide, such a polypeptide may also include afused polypeptide in which another polypeptide is fused at theN-terminus or the C-terminus of the polypeptide or fragment thereof. Afused polypeptide is produced by fusing a nucleic acid sequence (or aportion thereof) encoding one polypeptide to a nucleic acid sequence (ora portion thereof) encoding another polypeptide. Techniques forproducing fusion polypeptides are known in the art, and include,ligating the coding sequences encoding the polypeptides so that they arein frame and expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct may besynonymous with the term expression cassette when the nucleic acidconstruct contains all the control sequences required for expression ofa coding sequence. The term “coding sequence” is defined herein as asequence which is transcribed into mRNA and translated into a biologicalsubstance of interest when placed under the control of the belowmentioned control sequences. The boundaries of the coding sequence aregenerally determined by a translation start codon ATG at the 5′-terminusand a translation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

An isolated nucleic acid sequence directing synthesis of a biologicalsubstance may be manipulated in a variety of ways to provide forexpression of the biological substance. Manipulation of the nucleic acidsequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector or Bacillus host cell. Thetechniques for modifying nucleic acid sequences utilizing cloningmethods are well known in the art.

A nucleic acid construct comprising a nucleic acid sequence directingsynthesis of a biological substance may be operably linked to one ormore control sequences capable of directing the expression of the codingsequence in a mutant of a Bacillus cell under conditions compatible withthe control sequences.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for expression of the codingsequence of a nucleic acid sequence.

Each control sequence may be native or foreign to the nucleic acidsequence directing synthesis of the biological substance. Such controlsequences include, but are not limited to, a leader, a promoter, asignal sequence, and a transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence directing synthesis of a biological substance.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a Bacillus cell for expression ofthe nucleic acid sequence. The promoter sequence contains transcriptioncontrol sequences which mediate the expression of the biologicalsubstance. The promoter may be any nucleic acid sequence which showstranscriptional activity in the Bacillus cell of choice and may beobtained from genes directing synthesis of extracellular orintracellular biological substances either homologous or heterologous tothe Bacillus cell. Examples of suitable promoters for directing thetranscription of the nucleic acid constructs of the present invention,especially in a Bacillus cell, are the promoters obtained from the E.coli lac operon, the Streptomyces coelicolor agarase gene (dagA), theBacillus subtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (VIIIa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75:3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80:21-25). Further promoters are described in “Usefulproteins from recombinant bacteria” in Scientific American, 1980,242:74-94; and in J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a Bacillus cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence directing synthesis of thebiological substance. Any terminator which is functional in the Bacilluscell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by theBacillus cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence directing synthesis of the biologicalsubstance. Any leader sequence which is functional in the Bacillus cellof choice may be used in the present invention.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of apolypeptide which can direct the expressed polypeptide into the cell'ssecretory pathway. The signal peptide coding region may be native to thepolypeptide or may be obtained from foreign sources. The 5′ end of thecoding sequence of the nucleic acid sequence may inherently contain asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region which encodes the secretedpolypeptide. Alternatively, the 5′ end of the coding sequence maycontain a signal peptide coding region which is foreign to that portionof the coding sequence which encodes the secreted polypeptide. Theforeign signal peptide coding region may be required where the codingsequence does not normally contain a signal peptide coding region.Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to obtainenhanced secretion of the polypeptide relative to the natural signalpeptide coding region normally associated with the coding sequence. Thesignal peptide coding region may be obtained from an amylase or aprotease gene from a Bacillus species. However, any signal peptidecoding region capable of directing the expressed polypeptide into thesecretory pathway of a Bacillus cell of choice may be used in thepresent invention.

An effective signal peptide coding region for Bacillus cells is thesignal peptide coding region obtained from the maltogenic amylase genefrom Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylasegene, the Bacillus licheniformis subtilisin gene, the Bacilluslicheniformis beta-lactamase gene, the Bacillus stearothermophilusneutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilisprsA gene. Further signal peptides are described by Simonen and Palva,1993, Microbiological Reviews 57:109-137.

In the methods of the present invention, a recombinant expression vectorcomprising a nucleic acid sequence, a promoter, and transcriptional andtranslational stop signals may be used for the recombinant production ofa polypeptide or other biological substance. The various nucleic acidand control sequences described above may be joined together to producea recombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence directing synthesis of the polypeptide or biologicalsubstance at such sites. Alternatively, the nucleic acid sequence may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression, and possiblysecretion.

The recombinant expression vector may be any vector which can beconveniently subjected to recombinant DNA procedures and can bring aboutthe expression of the nucleic acid sequence. The choice of the vectorwill typically depend on the compatibility of the vector with theBacillus cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the Bacillus cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. The vector system may be a single vector or plasmidor two or more vectors or plasmids which together contain the total DNAto be introduced into the genome of the Bacillus cell, or a transposon.

The vectors may be integrated into the Bacillus cell genome whenintroduced into a Bacillus cell. For integration, the vector may rely onthe nucleic acid sequence directing synthesis of the biologicalsubstance or any other element of the vector for stable integration ofthe vector into the genome by homologous recombination. Alternatively,the vector may contain additional nucleic acid sequences for directingintegration by homologous recombination into the genome of the Bacilluscell. The additional nucleic acid sequences enable the vector to beintegrated into the Bacillus cell genome at a precise location in thechromosome. To increase the likelihood of integration at a preciselocation, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 1,500 base pairs,preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500base pairs, which are highly homologous with the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the Bacillus cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleic acidsequences.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in theBacillus cell in question. Examples of bacterial origins of replicationare the origins of replication of plasmids pBR322, pUC19, pACYC177, andpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,and pAMR1 permitting replication in Bacillus. The origin of replicationmay be one having a mutation to make its function temperature-sensitivein the Bacillus cell (see, e.g., Ehrlich, 1978, Proceedings of theNational Academy of Sciences USA 75:1433).

More than one copy of a nucleic acid sequence directing synthesis of abiological substance of interest may be introduced into the Bacilluscell to amplify expression of the nucleic acid sequence. Stableamplification of the nucleic acid sequence can be obtained byintegrating at least one additional copy of the sequence into theBacillus cell genome using methods well known in the art and selectingfor transformants. A convenient method for achieving amplification ofgenomic DNA sequences is described in WO 94/14968.

The vectors preferably contain one or more selectable markers whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide resistance, resistance toheavy metals, prototrophy to auxotrophs, and the like. Examples ofbacterial selectable markers are the dal genes from Bacillus subtilis orBacillus licheniformis, or markers which confer antibiotic resistancesuch as ampicillin, kanamycin, erythromycin, chloramphenicol ortetracycline resistance. Furthermore, selection may be accomplished byco-transformation, e.g., as described in WO 91/09129, where theselectable marker is on a separate vector.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

The transformation of the Bacillus cell may, for instance, be effectedby protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizize, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278).

The present invention also relates to methods of obtaining a mutant of aparent Bacillus cell, comprising: (a) introducing into the Bacillus cella first nucleic acid sequence comprising a modification of at least oneof the genes cypX and yvmC, which are involved in the production of ared pigment; and (b) identifying the mutant cell from step (a)comprising the modified nucleic acid sequence, wherein the mutant cellis deficient in the production of the red pigment.

The present invention further relates to mutants of a parent Bacilluscell, comprising a first nucleic acid sequence directing synthesis of aheterologous biological substance and a second nucleic acid sequencecomprising a modification of at least one of the genes cypX and yvmC,which are involved in the production of a red pigment, wherein themutant cell produces less of the red pigment than the parent Bacilluscell when cultivated under the same conditions.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

All primers and oligos were supplied by MWG Biotech, Inc., High Point,N.C.

Bacillus subtilis strains were made competent using the method describedby Anagnostopoulos and Spizizen, 1961, Journal of Bacteriology 81:741-746.

DNA sequencing was conducted with an ABI 3700 Sequencing (AppliedBiosystems, Inc., Foster City, Calif.).

Example 1 Identification of the cypX-yvmC and yvmB-yvmA Operons UsingDNA Microarrays

Bacillus subtilis strain RB128 is a Bacillus subtilis A164Δ5 strain(Bacillus subtilis ATCC 6051A deleted at the spoIIAC, aprE, nprE, amyE,and srfC genes) obtained according to the methods of U.S. Pat. No.5,891,701. Bacillus subtilis strain RB128 contains a heterologous geneencoding a Bacillus maltogenic amylase. Bacillus subtilis strain BRG1was obtained by N-methyl-N′-nitrosoguanidine (NTG) mutagenesis ofBacillus subtilis RB128 according to the following protocol. Bacillussubtilis RB128 cells grown to log phase were treated with threeconcentrations of N-methyl-N′-nitrosoguanidine (NTG): 0.26 mg/ml, 0.53mg/ml, and 1.06 mg/ml yielding percent kills of 98.2%, 99.5%, and 99.9%,respectively. One hundred microliters of each treatment was outgrown 6times in 1 ml aliquots in 24 well plates. The outgrowths were preservedin 10% glycerol and frozen at −80° C. The library size was approximately15500, 4200, and 250 mutants for each treatment, respectively. TheBacillus subtilis BRG1 mutant was isolated from the 0.26 mg/ml NTGtreatment Bacillus subtilis strains RB128 and BRG1 were cultivated for48 hours at 40-41° C., pH 7±0.2 in 1.5 liters of medium composed perliter of 50 g of hydrolyzed protein, 6.5 g of KH₂PO₄, 4.5 of Na₂HPO₄,3.0 g of (NH₄)₂SO₄, 2.0 g of Na₃-citrate-2H₂0, 3.0 g of MgSO₄, 0.15 mgof biotin, 0.5 g of CaCl₂-2H₂O, and trace metals. The fermentations werefed at a maximum rate of 8 g of saccharide per liter per hour. Thecultures were sparged with air at 1 to 2 liters per minute and agitatedat 1300 rpm. The whole broth color of Bacillus subtilis strain BRG1 waslight brown compared to the whole broth of Bacillus subtilis strainRB128 which was dark brown. Red pigment was visible in the cellularpellet of the whole broth from Bacillus subtilis strain RB128, while nored pigment was observed in the Bacillus subtilis strain BRG1 cellularpellet.

Total cellular RNA was obtained from 6, 12, 24, 29 and 46 hour samples(10 ml) of the fermentations of Bacillus subtilis strains RB128 andBRG1. The RNA was obtained from cell pellets prepared from thefermentation samples stored at −80° C. For RNA preparation, the frozencell pellet was resuspended in 1 ml of diethylpyrocarbonate(DEPC)-treated water, and nine replicates were prepared using the FastRNA Blue kit (Bio101, Inc., Vista, Calif.). The replicates were thenpooled into one tube for preparation of cDNA probes.

Ten replicate cDNA targets per time point were prepared and hybridizedto Bacillus subtilis ORFs PCR fragment microarrays according to Berka etal., 2002, Molecular Microbiology 43: 1331-1345 and Kane et al., 2000,Nucleic Acids Research 28: 4552-4557. Bacillus subtilis strain RB128cDNA was labeled with Cy5 (Amersham Corporation, Arlington Heights,Ill.) while Bacillus subtilis strain BRG1 cDNA was labeled with Cy3(Amersham Corporation, Arlington Heights, Ill.) according to theprocedure of Eisen and Burn, 1999, Methods in Enzymology 303: 179-205.Cy3 (a green fluorescent dye) and Cy5 (red fluorescent dye) reporterswere detected with solid state lasers operating at 532 nm and 632 nm,respectively. The arrays were scanned and formatted for analysis withQuantArray (PerkinElmer Lifesciences, Inc., Boston, Mass.) and importedinto GeneSpring (Silicon Genetics, Inc., Redwood City, Calif.) for finalanalysis. Statistical significance analysis of the replicate slides wasanalyzed with the SAM Excel add-in from Stanford University (Tusher etal., 2001, Proceedings of the National Academy of Sciences USA 98:5116-5121). The cypX-yvmC and yvmB-yvmA operons were identified aspotential sites involved in the formation of the red pigment,pulcherrimin (cypX: FIG. 1, SEQ ID NOs: 1 and 2, accession numberBG12580; yvmC: FIG. 2, SEQ ID NOs: 3 and 4, accession number BG14121;yvmB: FIG. 3, SEQ ID NOs: 5 and 6, accession number BG11018; and yvmA:FIG. 4, SEQ ID NOs: 7 and 8, accession number BG14120). The cypX-yvmCand yvmB-yvmA operons were consistently down-regulated in Bacillussubtilis strain BRG1 for the 12-46 hour time-points compared to Bacillussubtilis strain RB128.

A second microarray experiment was performed using two replicate cDNAtargets hybridized to Bacillus subtilis ORFs oligonucleotidesmicroarrays. The oligonucleotides were purchased from Compugen, Inc.,Jamesburg, N.J. and printed on poly-L-lysine coated slides at aconcentration of 10 μM to a density of four Bacillus subtilis genomesper slide as described by Berka et al., 2002, supra. Bacillus subtilisstrains RB128 and BRG1 cDNAs were labeled as described above. The arrayswere scanned and formatted for analysis using a GenePix 4000B scannerand GenePix Pro version 4.1 software (Axon Instruments, Inc., UnionCity, Calif.). Statistical significance analysis of the replicategenomes was analyzed with the SAM Excel add-in as above and significantgenes identified were imported to GeneSpring version 4.2. In this secondmicroarray experiment, only the cypX-yvmC operon was identified aspotential site involved in the formation of the red pigment.

Example 2 Construction of Bacillus subtilis Strain MaTa17

The cypX-yvmC and yvmB-yvmA operons were PCR amplified from Bacillussubtilis strain BRG1 as a single fragment using primers 1 and 2.

Primer 1: 5′-CATGGGAGAGACCTTTGG-3′ (SEQ ID NO: 9) Primer 2:5′-GTCGGTCTTCCATTTGC-3′ (SEQ ID NO: 10)

The amplification reactions (50 μl) were composed of 200 ng of Bacillussubtilis BRG1 chromosomal DNA, 0.4 μM each of primers 1 and 2, 200 μMeach of dATP, dCTP, dGTP, and dTTP, 1X Expand™ High Fidelity buffer with1.5 mM MgCl₂, and 2.6 units of Expand™ High Fidelity PCR System enzymemix (Roche Diagnostic Corporation, Indianapolis, Ind.). Bacillussubtilis BRG1 chromosomal DNA was obtained using a QIAGEN tip-20 column(QIAGEN, Inc., Valencia, Calif.) according to the manufacturer'sinstructions (Genomic DNA Handbook, QIAGEN, Inc., Valencia, Calif.,1999-2001, pp. 38-47). Amplification reactions were performed in aRoboCycler 40 thermacycler (Stratagene, Inc, La Jolla, Calif.)programmed for 1 cycle at 95° C. for 3 minutes; 10 cycles each at 95° C.for 1 minute, 58° C. for 1 minute, and 68° C. for 4 minutes; 20 cycleseach at 95° C. for 1 minute, 58° C. for 1 minute, 68° C. for 4 minutesplus 20 seconds per cycle, followed by 1 cycle at 72° C. for 7 minutes.Reaction products were analyzed by agarose gel electrophoresis using a0.8% agarose-25 mM Tris base-25 mM borate-0.5 mM disodium EDTA buffer(0.5×TBE) gel.

The resulting fragment comprising the cypX-yvmC and yvmB-yvmA operonswas cloned into pCR2.1 using the TA-TOPO Cloning Kit and transformedinto E. coli OneShot™ cells according to the manufacturer's instructions(Invitrogen, Inc., Carlsbad, Calif.). Transformants were selected onYeast-Tryptone (2×YT) agar plates supplemented with 100 μg of ampicillinper ml. Plasmid DNA from several transformants was isolated using QIAGENtip-20 columns according to the manufacturer's instructions and verifiedby DNA sequencing with M13 (−20) forward, M13 reverse and primers 3 to18 shown below. M13 (−20) forward and M13 reverse primers were obtainedfrom Invitrogen, Inc, Carlsbad, Calif. The resulting plasmid wasdesignated pMRT084 (FIG. 5).

Primer 3: 5′-CGACCACTGTATCTTGG-3′ (SEQ ID NO: 11) Primer 4:5′-GAGATGCCAAACAGTGC-3′ (SEQ ID NO: 12) Primer 5: 5′-CATGTCCATCGTGACG-3′(SEQ ID NO: 13) Primer 6: 5′-CAGGAGCATTTGATACG-3′ (SEQ ID NO: 14)Primer 7: 5′-CCTTCAGATGTGATCC-3′ (SEQ ID NO: 15) Primer 8:5′-GTGTTGACGTCAACTGC-3′ (SEQ ID NO: 16) Primer 9:5′-GTTCAGCCTTTCCTCTCG-3′ (SEQ ID NO: 17) Primer 10:5′-GCTACCTTCTTTCTTAGG-3′ (SEQ ID NO: 18) Primer 11:5′-CGTCAATATGATCTGTGC-3′ (SEQ ID NO: 19) Primer 12:5′-GGAAAGAAGGTCTGTGC-3′ (SEQ ID NO: 20) Primer 13:5′-CAGCTATCAGCTGACAG-3′ (SEQ ID NO: 21) Primer 14:5′-GCTCAGCTATGACATATTCC-3′ (SEQ ID NO: 22) Primer 15:5′-GATCGTCTTGATTACCG-3′ (SEQ ID NO: 23) Primer 16:5′-AGCTTTATCGGTGACG-3′ (SEQ ID NO: 24) Primer 17: 5′-TGAGCACGATTGCAGG-3′(SEQ ID NO: 25) Primer 18: 5′-CATTGCGGAGACATTGC-3′ (SEQ ID NO: 26)

DNA sequence comparison of the cypX-yvmC and yvmB-yvmA operons amplifiedfrom Bacillus subtilis BRG1 cloned into plasmid pMRT084 and thepublished sequence of Bacillus subtilis 168 (Kunst et al., 1997, Nature390:249-256) shows that these sequences are identical. In order tocreate a Bacillus subtilis strain deleted at these operons, plasmidpMRT084 was digested with BsgI to delete most of the cypX-yvmC andyvmB-yvmA operons, leaving about 500 bases at each end. The digestedBsgI DNA was treated with T4 DNA polymerase and shrimp alkalinephosphatase (SAP) according to the manufacturer's instructions (RocheDiagnostics Corporation, Indianapolis, Ind.). Plasmid pECC1 (Youngman etal., 1984, Plasmid 12: 1-9) was digested with SmaI. A fragment ofapproximately 5100 bp from pMRT084 and a fragment of approximately 1600bp fragment from pECC1 which contains the chloramphenicol resistancegene (cat) were isolated from a 0.8% agarose-0.5×TBE gel using theQIAquick DNA Extraction Kit (QIAGEN, Inc., Valencia, Calif.) accordingto the manufacturer's instructions, ligated, and used to transform E.coli XL1 Blue cells according to the manufacturer's instructions(Stratagene, Inc., La Jolla, Calif.). Transformants were selected on2×YT agar plates supplemented with 100 μg of ampicillin per ml.Transformants carrying the correct plasmid with most of the cypX-yvmCand yvmB-yvmA operons deleted were identified by PCR amplification usingprimers 19 and 20. PCR amplification was conducted in 50 μl reactionscomposed of 1 ng of plasmid DNA, 0.4 μM of each primer, 200 μM each ofdATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II (Applied Biosystems, Inc.,Foster City, Calif.) with 2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™DNA polymerase (Applied Biosystems, Inc., Foster City, Calif.). Thereactions were performed in a RoboCycler 40 thermacycler programmed for1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for7 minutes. The PCR product was visualized using a 0.8% agarose-0.5×TBEgel. This construct was designated pMRT086 (FIG. 6).

Primer 19: 5′-TAGACAATTGGAAGAGAAAAGAGATA-3′ (SEQ ID NO: 27) Primer 20:5′-CCGTCGCTATTGTAACCAGT-3′ (SEQ ID NO: 28)

Plasmid pMRT086 was linearized with ScaI and transformed into Bacillussubtilis RB128 competent cells in the presence of 0.2 μg ofchloramphenicol per ml. Transformants were selected on Tryptose bloodagar base (TBAB) plates containing 5 μg of chloramphenicol per ml, andgrown at 37° C. for 16 hours. Chromosomal DNA was prepared from severaltransformants using a QIAGEN tip-20 column according to themanufacturer's instructions. Chloramphenicol resistant colonies werescreened by PCR for deletion of the cypX-yvmC and yvmB-yvmA operons viaPCR using primers 3 and 19, 3 and 20, 4 and 19, and 4 and 20. PCRamplification was conducted in 50 μl reactions composed of 200 ng ofchromosomal DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1×PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycleseach at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1minute; and 1 cycle at 72° C. for 7 minutes. The PCR products werevisualized using a 0.8% agarose-0.5×TBE gel. The resulting Bacillussubtilis RB128 cypX-yvmC and yvmB-yvmA deleted strain was designatedBacillus subtilis MaTa17.

Bacillus subtilis MaTa17 was submitted for fermentation using the samemedium and conditions as described in Example 1. No observable redpigment was produced by Bacillus subtilis strain MaTa17 after 48 hours.Moreover, the second DNA microarray analysis in Example 1 identified thecypX-yvmC operon as the only operon involved in synthesis of the redpigment and Examples 3 and 4 below show that deletion of the cypX or theyvmC gene is necessary for elimination of the red pigment. Thus, inorder to test the usefulness of the elimination of the red pigment, thecypX gene was deleted in various Bacillus subtilis strains such asBacillus subtilis A164Δ5 (U.S. Pat. No. 5,891,701), Bacillus subtilisRB194, and Bacillus subtilis RB197 (WO 03/054163) as well as in otherBacillus strains as described herein where elimination of the redpigment would be beneficial in product recovery.

Example 3 Construction of Bacillus subtilis Strain A164Δ5ΔcypX

In order to verify the role the cypX gene plays in the synthesis of thered pigment, the cypX gene was deleted from Bacillus subtilis A164Δ5(U.S. Pat. No. 5,891,701). Plasmid pMRT122 (WO 03/054163) was used totransform Bacillus subtilis A164Δ5 competent cells. Transformants wereselected on TBAB-agar plates supplemented with 1 μg of erythromycin and25 μg of lincomycin per ml and incubated at 30° C. for 24-48 hours. Thedeleted cypX gene was introduced into the chromosome of Bacillussubtilis A164Δ5 via Campbell-type integration by incubating a freshlystreaked plate of Bacillus subtilis A164Δ5 (pMRT122) cells at 45° C. for16 hours and selecting for healthy growing colonies resulting inBacillus subtilis strain A165Δ5::pMRT122. Several of the healthy growingcolonies were inoculated into 1 ml of LB broth and incubated at 30° C.,250 rpm overnight. The cultured cells were serially passaged at leastthree times using 10 ml of cultured cells. After the last passage,cultured cells were streaked onto LB agar plates for isolation andincubated at 37° C., for 16 hours. Individual colonies were picked ontoLB agar and TBAB plates supplemented with 1 μg of erythromycin and 25 μgof lincomycin per ml in replicate fashion and grown at 37° C. for 16hours. Chromosomal DNA from potential integrants was isolated using theREDextract-N-Amp™ Plant PCR kits (Sigma Chemical Company, St. Louis,Mo.) as follows: Single Bacillus colonies were inoculated into 100 μl ofExtraction Solution (Sigma Chemical Company, St. Louis, Mo.), incubatedat 95° C. for 10 minutes, and then diluted with an equal volume ofDilution Solution (Sigma Chemical Company, St. Louis, Mo.). PCR wasperformed using 4 μl of extracted DNA in conjunction with theREDextract-N-Amp PCR Reaction Mix and primers 12 and 21 according to themanufacturer's instructions. PCR reactions were performed in aRoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 9minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute, 55° C. for 1minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes.The PCR product was visualized in a 0.8% agarose-0.5×TBE gel. Theresulting strain was designated Bacillus subtilis A164Δ5ΔcypX. Thepresence or loss of red pigment in Bacillus subtilis was visualized onSpizizen's minimal salts-agar (SMS) plates (Anagnostopoulos andSpizizen, 1961, supra) supplemented per liter with 0.5% sucrose, 0.15 mgof biotin, 24 mg of ferric sulfate, 9.6 mg of manganese sulfate, 3 mg ofcopper sulfate, 6 mg of zinc chloride, and 0.06% citric acid. Bacillussubtilis A164Δ5ΔcypX appeared to be colorless when compared to Bacillussubtilis A164Δ5.

Primer 21: 5′-CATGGGAGAGACCTTTGG-3′ (SEQ ID NO: 29)

Example 4 Construction of Strain Bacillus subtilis A164Δ5ΔyvmC

In order to validate if cypX and/or yvmC are responsible for thesynthesis of the red pigment, the yvmC gene was deleted, leaving thecypX gene intact. Plasmids pMRT074 (WO 03/054163) and pMRT084 weredigested with EcoRI and HindIII. A fragment of approximately 4300 bpfrom pMRT074 and a fragment of approximately 1700 bp from pMRT084 wereisolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNApurification kit according to the manufacturer's instructions, ligatedand used to transform Bacillus subtilis 16844 competent cells.Transformants were selected on TBAB-agar plates supplemented with 1 μgof erythromycin and 25 μg of lincomycin per ml and incubated at 30° C.for 24 hours. Transformants carrying the correct plasmid were identifiedon a 0.8% agarose-0.5×TBE gel by restriction analysis with DraI. Theresulting construct was designated pMRT126 (FIG. 7).

Plasmid pMRT126 was digested with Ecl136II/Eco47III to make a deletionin the yvmC gene, ligated and used to transform Bacillus subtilis 168Δ4.Transformants were selected on TBAB-agar plates supplemented with 1 μgof erythromycin and 25 μg of lincomycin per ml and incubated at 30° C.for 24 hours. Transformants carrying the correct plasmid were identifiedon a 2% agarose-0.5×TBE gel by restriction analysis with DraI. Theresulting plasmid was designated pMRT128 (FIG. 8).

Plasmid pMRT128 was used to transform Bacillus subtilis A164Δ5 competentcells.

Transformants were selected on TBAB-agar plates supplemented with 1 μgof erythromycin and 25 μg of lincomycin per ml and incubated at 30° C.for 24-48 hours. The deleted yvmC gene was introduced into thechromosome of Bacillus subtilis A164Δ5 via Campbell-type integration byincubating a freshly streaked plate of Bacillus subtilis A164Δ5(pMRT128) cells at 45° C. for 16 hours and selecting for healthy growingcolonies. Several of the healthy growing colonies were inoculated into 1ml of LB broth and incubated at 30° C., 250 rpm overnight. The culturedcells were serially passaged at least three times using 10 μl ofcultured cells. After the last passage, cultured cells were streakedonto LB agar plates for isolation and incubated at 37° C., for 16 hours.Individual colonies were picked onto LB agar, TBAB plates supplementedwith 1 μg of erythromycin and 25 μg of lincomycin per ml, and SMS platescontaining trace metals described in Example 3, in replicate fashion,and grown at 37° C. for 16-48 hours. Chromosomal DNA from erythromycinsensitive colonies was isolated using the REDextract-N-Amp™ Plant PCRkits as described in Example 3, and screened by PCR for the deleted yvmCgene with primers 7 and 10 using PCR cycling conditions described inExample 3. PCR products were visualized in a 0.8% agarose-0.5×TBE gel.The presence or loss of the red pigment in Bacillus subtilis wasvisualized on the Spizizen's minimal salts-agar (SMS) plates containingtrace metals. The yvmC-deleted strain appeared to be colorless whencompared to wild-type strains, and was designated Bacillus subtilisA164Δ5ΔyvmC.

Example 5 Fermentations of Bacillus subtilis Strains

Bacillus subtilis strains RB187, RB194 and RB197, constructed asdescribed in WO 03/054163, were cultivated in a 3 liter fermentorcontaining 1.5 liters of minimal salts medium composed per liter of 6.5g of KH₂PO₄, 4.5 g of Na₂HPO₄, 3.0 g of (NH₄)₂SO₄, 2.0 g of sodiumcitrate, 3.0 g of MgSO₄.H₂O, 0.15 g of biotin, 15 g of saccharide, 0.5 gof CaCl₂.2H₂O, and trace elements. The fermentation was fed withsaccharide at a rate of 2 g of saccharide/liter/hour. The cultures weresparged with air at 1 to 2 liters per minute and agitated at 1250 rpm.The fermentations were maintained at a pH of 7.0±0.2 and a temperatureof 32-37° C. The production of red pigment was visible by 12 hours inthe whole broth supernatant and cell pellet, and intensified for theremainder of the fermentation, up to 48 hours with Bacillus subtilisstrain RB187. No visible production of red pigment was observed withBacillus subtilis strains RB194 and RB197. Table 1 summarizes theresults for the strains evaluated for red pigment synthesis in thisinvention.

TABLE 1 Summary of strains evaluated for red pigment synthesis RedStrain Reference Gene deletion pigment Bacillus subtilis MaTa17 Example2 cypX, yvmC, yvmA No and yvmB Bacillus subtilis RB187 WO 03/054163 NoneYes Bacillus subtilis RB194 WO 03/054163 cypX, yvmC, yvmA No and yvmBBacillus subtilis RB197 WO 03/054163 cypX No Bacillus subtilis Example 3cypX No A164Δ5ΔcypX Bacillus subtilis Example 4 yvmC No A164Δ5ΔyvmCBacillus licheniformis Example 6 cypX No SJ1904ΔcypX

Example 6 Construction of strain Bacillus licheniformis SJ1904ΔcypX

The cypX gene from Bacillus licheniformis SJ1904 (U.S. Pat. No.5,733,753) was PCR amplified with primers 22 and 23.

Primer 22: 5′-GAATTCGCAGGAGGAACGAGTATG-3′ (SEQ ID NO: 30) Primer 23:5′-AAGCTTGAAGATCAGTGAGGCAGC-3′ (SEQ ID NO: 31)

The amplification reactions (50 μl) were composed of 200 ng of Bacilluslicheniformis SJ1904 chromosomal DNA, 0.4 μM each of primers 22 and 23,200 μM each of dATP, dCTP, dGTP, and dTTP, 1X Expand™ High Fidelitybuffer with 1.5 mM MgCl₂, and 2.6 units of Expand™ High Fidelity PCRSystem enzyme mix (Roche Diagnostic Corporation, Indianapolis, Ind.).Bacillus licheniformis SJ1904 chromosomal DNA was obtained using aQIAGEN tip-20 column according to the manufacturer's instructions.Amplification reactions were performed in a RoboCycler 40 thermacyclerprogrammed for 1 cycle at 94° C. for 2 minutes; 30 cycles each at 94° C.for 1 minute, 52° C. for 1 minute, and 68° C. for 2 minutes, followed by1 cycle at 72° C. for 7 minutes. Reaction products were analyzed byagarose gel electrophoresis using a 0.8% agarose-0.5×TBE gel. Theresulting fragment (approximately 1300 bp) comprising the cypX gene wascloned into pCR2.1 using the TA-TOPO Cloning Kit and used to transformE. coli OneShot™ cells according to the manufacturer's instructions.Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml. Plasmid DNA from several transformants wasisolated using QIAGEN tip-20 columns according to the manufacturer'sinstructions and verified by DNA sequencing with M13 (−20) forward andM13 reverse primers. The resulting plasmid was designated pMRT121 (FIG.9).

Plasmid pMRT121 was digested with NruI and PmlI to make a deletion inthe cypX gene leaving about 350 bp at each end, ligated, and used totransform E. coli XL1 Blue cells according to the manufacturer'sinstructions. Transformants were selected on 2×YT agar platessupplemented with 100 μg of ampicillin per ml and incubated at 37° C.for 16 hours. Transformants carrying the correct plasmid were identifiedon a 2% agarose-0.5×TBE gel by restriction analysis with DraI. Theresulting plasmid was named pMRT123 (FIG. 10).

Plasmids pMRT074 and pMRT123 were digested with EcoRI and HindIII. Afragment of approximately 700 bp from pMRT123 and a fragment ofapproximately 4300 bp from pMRT074 were isolated from a 0.8%agarose-0.5×TBE gel using a QIAquick DNA purification kit according tothe manufacturer's instructions, ligated, and used to transform Bacillussubtilis A168Δ4 competent cells. Transformants were selected onTBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg oflincomycin per ml and incubated at 30° C. for 24 hours. Transformantscarrying the correct plasmid were identified on a 2% agarose-0.5×TBE gelby restriction analysis with DraI. The resulting construct wasdesignated pMRT124 (FIG. 11).

Plasmid pMRT124 was used to transform Bacillus licheniformis SJ1904electrocompetent cells according to the method described by Xue et al.,1999, Journal of Microbiological Methods 34:183-191. Afterelectroporation, cells were incubated in LBSM medium (Luria-Bertanimedium containing 0.5 M sorbitol and 0.38 M mannitol) supplemented with0.2 μg/ml erythromycin for 2.5 to 3 hours, plated on TBAB-agar platessupplemented with 1 μg of erythromycin and 25 mg of lincomycin per mland incubated at 30° C. for 24-48 hours. The deleted cypX gene inplasmid pMRT124 was introduced into the B. licheniformis SJ1904chromosome via Campbell-type integration by incubating a freshlystreaked plate of Bacillus licheniformis A164Δ5 (pMRT124) cells at 50 Cfor 16 hours and selecting for healthy growing colonies. Several of thehealthy growing colonies were inoculated into 1 ml of LB broth andincubated at 30° C., 250 rpm overnight. The cultured cells were seriallypassaged at least three times using 10 μl of cultured cells. After thelast passage, cultured cells were streaked onto LB agar plates forisolation and incubated at 37° C., for 16 hours. Individual colonieswere picked onto LB agar and TBAB plates supplemented with 1 mg oferythromycin and 25 μg of lincomycin per ml in replicate fashion andgrown at 37° C. for 16 hours. Chromosomal DNA from erythromycinsensitive colonies was isolated using the REDextract-N-Amp™ Plant PCRkits as described in Example 3, and screened by PCR for the deleted cypXgene with primers 22 and 23 using PCR cycling conditions are describedin Example 3. PCR products were visualized in a 0.8% agarose-0.5×TBEgel. The resulting strain was designated Bacillus licheniformisSJ1904ΔcypX. The presence or loss of the red pigment formation byBacillus licheniformis was visualized by streaking Bacilluslicheniformis SJ1904 and B. licheniformis SJ1904ΔcypX side-by-side onthe Spizizen's minimal salts-agar (SMS) plates supplemented with tracemetals (Example 3). Plates were incubated at 37° C. for 48 hours. ThecypX-deleted strain appeared to be colorless when compared to thecontrol strain, indicating that loss of red pigment formation wasaccomplished by deleting the cypX gene.

Example 7 Isolation of Red Pigment from RB187 Supernatant

The red pigment found in the broth of strain Bacillus subtilis RB187 wasisolated by adjusting 40 ml of supernatant to pH 1.5 with 6 N HCl. Theacidified broth was incubated at 94° C. for 30 minutes and the pigmentwas pelleted by centrifugation in a SORVALL 6000B centrifuge at 2500rpm, 4° C. for 20 minutes (SORVALL, Inc., Newtown, Conn.). The redpellet was washed by centrifugation three times with 20 ml HPLC-gradewater, dissolved in 10 ml alkaline methanol and recovered byacidification to pH 1.5 in the presence of excess ferric chloride asdescribed by Canale-Parola, 1963, Archiv für Mikrobiologie 46: 414-427.Spectral analysis of the red pigment in 2 M NaOH from 600 nm to 200 nmyields an absorption spectrum with peaks at 242 nm, 280 nm and 242 nm.This UV-visible spectrum of the purified pigment is similar to thepulcherrimin absorption spectrum found by Canale-Parola. Collectively,the solubility in alkaline methanol, insolubility in acid, andcharacteristic absorbance spectrum strongly suggest that the red pigmentis pulcherrimin.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of producing a heterologous protein,comprising: (a) cultivating a mutant Bacillus cell transformed with anucleic acid construct comprising a nucleic acid directing synthesis ofthe heterologous protein in a medium suitable for the production of theheterologous protein, wherein the mutant cell comprises a deletionmutation in a cypX gene, a yvmC gene, or both genes, in which thedeletion mutation(s) renders the cell deficient in red pigment comparedto a wild-type Bacillus cell comprising the cypX gene, the yvmC gene, orboth genes, wherein the cypX gene comprises a nucleic acid sequencehaving at least 90%, at least 95%, or 100% homology to SEQ ID NO: 1 andthe yvmC gene comprises a nucleic acid sequence having at least 90%, atleast 95%, or 100% homology to SEQ ID NO: 7; and (b) recovering theheterologous protein from the cultivation medium.
 2. The method of claim1, wherein the heterologous protein is involved in the biosynthesis of abiopolymer.
 3. The method of claim 1, wherein the heterologous proteinis involved in the biosynthesis of a metabolite.
 4. The method of claim1, wherein the mutant cell produces no detectable red pigment comparedto the parent Bacillus cell when cultured under identical conditions. 5.The method of claim 1, wherein the mutant cell is further deficient inthe production of one or more selected from the group consisting ofprotease, amylase, and surfactin.
 6. The method of claim 1, wherein themutant Bacillus cell does not produce spores.
 7. An isolated mutantBacillus cell, comprising a deletion mutation in a cypX gene, a yvmCgene, or both genes, in which the deletion mutation(s) renders the celldeficient in red pigment compared to a wild-type Bacillus cellcomprising the cypX gene, the yvmC gene, or both genes, transformed witha nucleic acid construct comprising a nucleic acid directing synthesisof the heterologous protein, wherein the cypX gene comprises a nucleicacid sequence having at least 90%, at least 95%, or 100% homology to SEQID NO: 1 and the yvmC gene comprises a nucleic acid sequence having atleast 90%, at least 95%, or 100% homology to SEQ ID NO:
 7. 8. The mutantcell of claim 7, wherein the heterologous protein is involved in thebiosynthesis of a biopolymer.
 9. The mutant cell of claim 7, wherein theheterologous protein is involved in the biosynthesis of a metabolite.10. The mutant cell of claim 7, which produces no detectable red pigmentcompared to the parent Bacillus cell when cultured under identicalconditions.
 11. The mutant cell of claim 7, which is further deficientin the production of one or more selected from the group consisting ofprotease, amylase, and surfactin.
 12. The mutant cell of claim 7, whichdoes not produce spores.
 13. A method of producing an isolated mutantBacillus cell, comprising: making a deletion mutation in a cypX gene, ayvmC gene, or both genes, of a Bacillus cell, in which the deletionmutation(s) renders the cell deficient in red pigment compared to awild-type Bacillus cell comprising the cypX gene, the yvmC gene, or bothgenes, and transforming the cell with a nucleic acid constructcomprising a nucleic acid directing synthesis of a heterologous protein,wherein the cypX gene comprises a nucleic acid sequence having at least90%, at least 95%, or 100% homology to SEQ ID NO: 1 and the yvmC genecomprises a nucleic acid sequence having at least 90%, at least 95%, or100% homology to SEQ ID NO:
 7. 14. The method of claim 13, wherein theheterologous protein is involved in the biosynthesis of a biopolymer.15. The method of claim 13, wherein the heterologous protein is involvedin the biosynthesis of a metabolite.
 16. The method of claim 13, whereinthe mutant cell produces no detectable red pigment when compared to theparent Bacillus cell when cultured under identical conditions.
 17. Themethod of claim 13, wherein the mutant Bacillus cell is furtherdeficient in the production of one or more selected from the groupconsisting of protease, amylase, and surfactin.
 18. The method of claim13, wherein the mutant Bacillus cell does not produce spores.